Virtually every area of the biomedical sciences needs to determine the presence, structure, and function of particular analytes that participate in chemical and biological interactions. The needs range from the basic scientific research lab, where biochemical pathways are being mapped and correlated to disease processes, to clinical diagnostics, where patients are routinely monitored for levels of clinically relevant analytes. Other areas include pharmaceutical research, military applications, veterinary, food, and environmental applications. In all of these cases, the presence, quantity, and structure activity relationships of a specific analyte or group of analytes needs to be determined.
Numerous methodologies have been developed to meet this need. The methods include enzyme-linked immunosorbent assays (ELISA), radio-immunoassays (RIA), numerous fluorescence assays, mass spectrometry, colorimetric assays, gel electrophoresis, as well as a host of more specialized assays. Most of the assay techniques require specialized preparations such as chemically attaching a label or purifying and amplifying a sample to be tested. Generally, an interaction between two or more molecules is monitored via a detectable signal relating to the interaction. Typically a label conjugated to either a ligand or anti-ligand of interest generates the signal. Physical or chemical effects produce detectable signals. The signals may include radioactivity, fluorescence, chemiluminescence, phosphorescence, and enzymatic activity. Spectrophotometric, radiometric, or optical tracking methods can be used to detect many labels.
Unfortunately, in many cases it is difficult or even impossible to label one or all of the molecules needed for a particular assay. The presence of a label may interrupt molecular interaction or otherwise make the molecular recognition between two molecules not function for many reasons including steric effects. In addition, none of these labeling approaches can determine the exact nature of the interaction. Active site binding to a receptor, for example, is indistinguishable from non-active site binding, and thus no functional information is obtained from the present detection methodologies. A method to detect interactions that eliminates the need for the label and that yields functional information would greatly improve upon the above mentioned approaches.
Detection technology is commercially very important. The biomedical industry relies on tests for a variety of water-based or fluid-based physiological systems to evaluate protein-protein interactions, drug-protein interactions, small molecule binding, enzymatic reactions, and to evaluate other compounds of interest. Ideally, the technology should not require highly specific probes, such as specific antibodies. The assay should operate by measuring the native properties of molecules and would not require additional label(s) or tracer(s) to detect a binding event. In many applications, the assay should be miniaturizable and handle samples in parallel, so that complex biochemical pathways can be mapped out, or extremely small and numerous quantities of compounds can be used in drug screening protocols. For many applications, the assay should monitor in real time, a complex series of reactions, such that accurate kinetics and structure-activity relationships can be obtained almost immediately.
Vibrational spectroscopy overcomes limitations in this field and is a well established, non-destructive, analytical tool that can reveal much information about molecular interactions. Infrared spectroscopy involves the absorption of electromagnetic radiation generally between 0.770–1000 microns (12,900–10 cm−1), which represent energies on the order of those found in the vibrational transitions of molecular species. Variations in the positions, widths, and strengths of these modes with composition and structure allow identification of molecular species. One advantage of infrared spectroscopy is that virtually any sample, in virtually any state, can be studied without the use of a separate label. Liquids, solutions, pastes, powders, films, fibers, gases, and surfaces can be examined by a judicious choice of sampling techniques. Biological systems such as proteins, peptides, lipids, bio-membranes, carbohydrates, pharmaceuticals, foods, and both plant and animal tissues have been characterized with infrared spectroscopy as reviewed by B. Stuart in Modem Infrared Spectroscopy (Wiley and Sons) Chichester (1996) and in Biological applications of Infrared Spectroscopy (Wiley and Sons) Chichester 1997.
The availability of high-resolution infrared spectrometers has led to time resolved investigations of chemical and biological interactions, which include cell cycle investigations (e.g. H. Y. Holman, M. C. Martin, E. A. Blakely, K. Bjornstad, W. R. McKinney, Biolpolmers (Biospectroscopy) 2000, 57, 329–335), protein-protein interactions (e.g. R. Barbucci, A. Magnani, C. Roncolini, S. Silvestri, Biopolymers 1991, 31, 827–834), polymerization studies (e.g. P. K. Aldridge, J. J. Kelly, J. B. Callis, D. H. Burns, Anal. Chem. 1993, 65, 3581–3585), and solid-phase organic reactions (e.g. B. Yan, J. B. Fell, G. Kumaravel, J. Org. Chem. 1996, 61, 7467–7472). These investigations traditionally have been restricted to one-at-a-time measurements because of single detectors used for conventional infrared spectrometers. Autosamplers have been introduced, which move either the optical path over the samples or a number of samples through the optical path sequentially using a computer controlled system. See for example, http://www.optics.bruker.com/pages/products/BIO/hts-xt.htm; http://www.piketech.com/catalog/pdfs/autotrns.pdf. However, data collection mostly remains serial, making kinetic investigations cumbersome, if not impossible, for a large number of reactions.
Serial one-sample investigations also have been addressed by detector arrays such as a focal plane array which uses infrared spectral imaging for remote sensing, as described by R. Beer Remote sensing by Fourier transform spectrometry (Chemical Analysis v. 120) 1992, Wiley and Sons, New York. Spectral imaging also has been coupled to use of an infrared microscope (See for example U.S. Pat. No. 5,377,003 and references therein and B. Foster, American Laboratory Feb. 21–29, 1997, and P. J. Treado, M. D. Morris, Applied Spectroscopy Reviews 1994, 29(1), 1–38) for imaging studies of plant and animal tissue, polymer dissolution, and polymer liquid crystals. These single sample procedures purport to collect spatially correlated spectral information (i.e. a spectral image). More recently, infrared spectra have been made from multiple samples in parallel and is particularly advantageous for high throughput screening of the large numbers of chemical products in combinatorial investigations. For example, published patent application WO 98/15813 describes the use of parallel detection infrared spectroscopy for monitoring catalytic reactions and other applications of high resolution imagery for “single samples” (see http://www.spectraldimensions.com). This patent application describes measurements primarily in the transmission mode but unfortunately, lacks information needed to make a realistic system. For example, the discussion and figures of sample holders for the transmission measurements do not explain how to transfer samples into a sample array. The assumption presented that a robot would fill sample arrays and then a human would have to “cap” the arrays with an infrared transparent “top” is impractical for an automated high-throughput screening environment. Thus, this described system at best appears limited to the high-resolution imagery on “single-samples.”
Unfortunately, these systems suffer sensitivity and/or speed limitations. One reason is that as sample number increases the actual size of a single decreases. The number of photons that can interact with the sample in a short time to generate a meaningful signal decreases dramatically and generally limits both sensitivity and speed. A solution to this problem would open up new areas of discovery and would be particularly important in the burgeoning field of combinatorial chemistry, which require rapid assay of huge numbers of very tiny samples.